Construção e seleção de uma biblioteca combinatorial de anticorpos contra herpesvirus bovino tipo 1

• Autor(a) principal: Japolla, Greice
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/4215
• topic: Imunoglobulinas; Fragmento scFv; Immunoglobulins; ScFv fragment; Phage display; MEDICINA VETERINARIA PREVENTIVA::EPIDEMIOLOGIA ANIMAL

Bovine herpesvirus type 1 ( BHV - 1 ) is recognized as an important pathogen of economic losses in cattle , these animals causing diseases known as infectious bovine rhinotracheitis ( IBR ) , infectious pustular vulvovaginitis , infectious balanoposthitis and neurological disorders . For effective control of these diseases, the correct diagnosis is necessary, but none of the available tests enables a quick result made the field. Considering this, the aim of this study was to construct a combinatorial antibody library, for it to be used in the future development of new diagnostic approaches . Two breed White Leghorn chickens were immunized with 105.5 TCID50/ml of BoHV - 1, birds were necropsied , their spleens removed for total RNA extraction , cDNA synthesis , amplification of gene fragments encoding the light chain ( VL ) and heavy ( VH ) and production of scFv ( v) fragments. These fragments were cloned into vectors fagomidiais expressed as fusion proteins on filamentous phage and amplified by infection of E. coli. Selection of viral particles ( fused scFv) binding to the BHV -1 ( biopanning ) by six cycles were performed. The affinity of the scFv antibody library BHV -1 observed in ELISA shows that the produced fragments are reactive to HIV, the use of such antibodies in the development of new diagnostic platforms is possible . The sequencing results showed a reduction of variability in comparison to the dot blot previously performed, and a desirable feature of this process , however, it was possible to sequence the clones efficiently, it has been found , therefore, a need to further analyze the shape of results